Relatively little regarding the function of the viral protein R (Vpr) has been reported since the demonstration that the small open reading frame within HIV-1 designated R encodes a 15 kd protein. Wong-Staal, et al., AIDS Res. Hum. Retroviruses, 1987, 3, 33-39. The vpr open reading frame is conserved within all genomes of HIV-1 and HIV-2 and within most, if not all, simian immunodeficiency virus (SIV) genomes. Vpr is immunogenic in vivo and most if not all HIV+ individuals makes antibodies that can react with eukaryotically produced Vpr protein.
The progression from HIV infection to AIDS is in large part determined by the effects of HIV on the cells that it infects, including CD4− T lymphocytes and macrophages. On the other hand, cell activation, differentiation and proliferation are in turn thought to regulate HIV infection and replication in T cells and macrophages. Gallo, et al., Science, 1984, 224, 500; Levy, et al., Science, 1984, 225, 840; Zack. et al., Science, 1988, 240, 1026; Griffin, et al., Nature, 1988, 339, 70; Valentin, et al., J. AIDS, 1991, 4, 751; Rich, et al., J. Clin. Invest., 1992, 89, 176; and Schuitemaker, et al., J. Virol., 1992, 66, 1354. Cell division per se may not be required since HIV and other lentiviruses can replicate in nonproliferating, terminally differentiated macrophages and growth-arrested T lymphocytes. Rose, et al., Am. Rev. Respir. Dis., 1986, 143, 850; Salahuddin, et al., Blood, 1986, 68, 281; and Li, et al., J. Virol., 1993, 67, 3969. The ability of lentiviruses, including HIV, to replicate in nonproliferating cells, particularly in macrophages, is believed to be unique among retroviruses and it may be significant that several lentiviruses contain a vpr-like gene. Myers, et al. AIDS Res. Hum. Retrovir., 1992, 8, 373. HIV infection of myeloid cell lines can result in a more differentiated phenotype and increase the expression of factors such as NF-KB which are necessary for HIV replication. Roulston, et al., J. Exp. Med. 1992, 175, 751; and Chantal Petit, et al., J. Clin. Invest., 1987, 79, 1883.
The most evidence for the function of the Vpr protein comes from several studies reporting the activities of HIV strains that have mutations in the vpr gene. It has been reported that mutations in the vpr gene results in a decrease in the replication and cytopathogenicity of HIV-1, HIV-2, and SIV in primary CD4−T lymphocytes and transformed T cell lines (Ogawa, et al., J. Virol., 1989, 63, 4110-4114; Shibata, et al. J. Med. Primatol., 1990a, 19, 217-225; Shibata, et al., J. Virol., 1990b, 64, 742-747 and Westervelt, et al., J. Virol., 1992, 66, 3925), although others have reported mutated vpr gene had no effect on replication (Dedera, et al., Virol., 1989, 63, 3205-3208). Interestingly HIV-2 mutated for vpr has been reported unable to infect primary monocyte/macrophages. Hattori, et al., Proc. Natl. Acad. Sci. USA, 1990, 87, 8080-8084. Transactivation of the HIV long terminal repeat and heterologous promoters by HIV is increased about 3-fold in wild-type versus vpr-negative HIV-1, though the mechanism through which Vpr may transactivate transcription is unknown and may be indirect. Cohen, et al., J. Acquir. Immune Defic. Syndr., 1990b, 3, 11-18. The relationship between the effects of Vpr on promoter activity and viral infectivity is not clear. Vpr protein is incorporated into the viral particle, and this finding has led to the proposition that Vpr functions early in infection, following virus penetration and uncoating, and that Vpr may interact with cellular regulatory mechanisms important in the establishment of infection. Cohen, et al., J. Virol., 1990a, 64, 3097-3099; Yu, et al., J. Virol., 1990, 64, 5688-5693; and, Yuan, et al., AIDS Res. Hum. Retroviruses, 1990, 6, 1265-1271.
The vpr gene of HIV-1 has been shown to induce cellular growth inhibition and differentiation in tumor lines of intermediate differentiation in vitro Levy, et al., Cell, 1993, 72, 541. Since Vpr protein originates within viral particles. Vpr may play a role in establishing productive infection. In addition, several important possibly interrelated functions have been identified for HIV-1 Vpr. These include import of reverse transcription complex into the nucleus of non-dividing cells, cellular differentiation, cell cycle arrest at the G2/M phase, and enhancement of HIV-1 replication.
HIV-1 Vpr is required to import the viral preintegration complex into the nucleus of non-dividing cells (Heinzinger, et al., Proc. Natl. Acad. Sci. USA, 1994, 91, 7311-7315; and Fletcher, et al., EMBO., 1996, 15, 6155-6165) and it enhances viral replication in monocyte cell lines (Balotta, et al., J. Virol., 1993, 67, 4409-4414; Balliet, et al., Virology, 1994, 200, 623-631; and Connor, et al., Virology, 1995, 206, 935-944). Vpr localizes to the nucleus and induces cellular differentiation subsequently arresting cells at the G2/M phase of the cell cycle. Lu, et al., J. Virol., 1993, 67, 6542-6550; Mahalingam, et al., Virology, 1995, 212, 331-339; DiMarzio, et. al., J. Virol., 1995, 69, 7909-7916; Levy, et al., Cell, 1993, 72, 541-550; Rogel, et al., J. Virol., 1995, 69, 882-888; Jowett, et al., J. Virol., 1995, 69, 6304-6313; Mahalingam, et al., DNA Cell Biol., 1997, 16, 137-153. Mutational analysis suggests that the functions of this 96 amino acid Vpr protein are mediated through interactions with appropriate cellular cofactor(s). Zhao, et al., J. Biol. Chem., 199, 269, 15577-15582; Refaeli, et al., Proc. Natl. Acad. Sci. USA, 1995, 92, 3621-3625; He, et al., J. Virol., 1995, 69, 6705-6711; Re, et al., J. Virol., 1995, 69, 6859-6864.
There is a need to identify novel compounds which inhibit HIV replication. Specifically, safe and effective compounds are sought which reduce replication by interfering with particular molecular signals mediated by Vpr. Likewise, safe and effective compounds are sought which interfere with the cofactor with which Vpr interacts, which is an essential component of the cell cycle cascade. Moreover, there is a need to identify the co-factor and target it in methods of modulating the cell cycle. There is a need for compounds and methods for inhibiting the progression of the cell cycle from G2 to M phase in cells whose proliferation is undesirable such as hyperproliferating cells.